This project involves a study of the regulation of histidine catabolism in Pseudomonas putida, emphasizing the catalytic and regulatory properties of the first two enzymes, histidase and urocanase, and factors which affect their synthesis. The key enzyme histidase is being investigated from three approaches. The first is aimed at elucidating the post-translational events responsible for formation of the dehydroalanine prosthetic group, namely what enzymic reactions give rise to dehydroalanine residues in histidase, by an examination of histidase precursor species which are believed to be accumulated in mutants blocked in the post-translational modification steps. The second approach is to establish in what way dehydroalanine is associated with the histidase protein so that its stability is maintained. This is postulated to be by the formation of an addition compound with NAD tightly bound to histidase. Thirdly, purine nucleotides are effective regulators of histidase activity and to obtain more insight into how this regulation occurs, studies on nucleotide binding and on oligomeric state, as influenced by nucleotides, are being undertaken. The second enzyme, urocanase, is known to contain NAD ion as a catalytically important coenzyme. It has been shown that NADH is not an intermediate in the reaction, and current evidence suggests NAD functions by formation of an adduct with the imidazole group of substrates and substrate analogues. These adducts are being characterized as to their precise structure. An active site SH group on urocanase is also being studied to determine its role catalysis. Also planned is a study to determine the extent to which histidyl-tRNA his levels influence the expression of the genes for histidine utilization. This study will involve measuring histidyl-tRNA levels in mutants with altered amino acyl tRNA synthetases under conditions where the cells are induced for the hut enzymes, to permit some assessment of the role of histidyl-tRNA as a positive regulator for the hut operon.